Modified silk fibroin artificial blood vessel as well as preparation method and application thereof

Abstract

The invention discloses a modified silk fibroin artificial blood vessel and a preparation method and application thereof.The modified silk fibroin artificial blood vessel comprises a modified silk fibroin composite scaffold and a polyurethane layer, the polyurethane layer is arranged on the modified silk fibroin composite scaffold through electrostatic spinning, and the polyurethane layer is prepared from polyurethane, the modified silk fibroin composite scaffold is prepared from the following raw materials: modified silk fibroin, arginine-glycine-aspartic acid polypeptide modified polycaprolactone, bacterial cellulose, a growth factor and glutaraldehyde. The modified silk fibroin is in bridge connection with the arginine-glycine-aspartic acid polypeptide modified polycaprolactone and is loaded with the growth factor. The comprehensive biocompatibility of the artificial blood vessel can be improved, thrombosis and intimal hyperplasia are effectively inhibited, and therefore the long-term patency rate of the small-caliber artificial blood vessel is greatly increased.

Description

Technical Field

[0001] This application relates to the field of medical materials technology, and in particular to a modified silk fibroin artificial blood vessel, its preparation method, and its application. Background Technology

[0002] With the global incidence of cardiovascular and cerebrovascular diseases continuing to rise, vascular stenosis or occlusion caused by atherosclerosis, embolism, and other factors has become a major clinical challenge threatening public health. For severe vascular lesions, surgical transplantation is the primary treatment. Although autologous blood vessels (such as the patient's internal mammary artery or great saphenous vein) are considered the "gold standard" for transplantation, their availability is limited, and they face inherent limitations such as secondary trauma to the donor site, the risk of complications, and poor vessel quality in some patients. Therefore, developing high-performance artificial blood vessels as reliable alternative grafts is crucial.

[0003] Currently, commercially available traditional artificial blood vessels are mainly based on two types of materials: braided polyester and expanded polytetrafluoroethylene (ePTFE). These materials have achieved relatively satisfactory results in the replacement of large and medium diameter (usually referring to an inner diameter greater than 6 mm) blood vessels (such as aortic surgery). However, in the replacement of small diameter (inner diameter less than 6 mm) blood vessels (such as coronary arteries, infrakal arteries, etc.), these artificial blood vessels still generally face the problem of early postoperative thrombosis and intimal hyperplasia, resulting in low long-term graft patency. Summary of the Invention

[0004] To improve thrombosis and patency of artificial blood vessels, this application provides a modified silk fibroin artificial blood vessel, its preparation method, and its application.

[0005] In a first aspect, this application provides a modified silk fibroin artificial blood vessel, employing the following technical solution: A modified silk fibroin artificial blood vessel includes a modified silk fibroin composite scaffold and a polyurethane layer. The polyurethane layer is electrospun onto the modified silk fibroin composite scaffold. The raw materials for preparing the polyurethane layer include polyurethane. The raw materials for preparing the modified silk fibroin composite scaffold include modified silk fibroin, arginine-glycine-aspartic acid peptide-modified polycaprolactone, bacterial cellulose, growth factors, and glutaraldehyde. The modified silk fibroin bridges the arginine-glycine-aspartic acid peptide-modified polycaprolactone and is loaded with growth factors.

[0006] By employing the above-mentioned technical solution, this application constructs a biomimetic double-layer artificial blood vessel. The outer layer is a polyurethane layer, fabricated through electrospinning, which effectively improves the mechanical strength, elasticity, and fatigue resistance of the artificial blood vessel, meeting the mechanical requirements after implantation. The inner layer is a modified silk fibroin composite scaffold, directly in contact with blood. The modified silk fibroin and bacterial cellulose provide a good biocompatibility substrate, which is conducive to endothelial cell adhesion and growth, thereby reducing abnormal platelet adhesion and activation and decreasing the risk of early thrombosis. The arginine-glycine-aspartic acid peptide in the composite scaffold can specifically recognize and anchor endothelial (progenitor) cells to the inner surface of the artificial blood vessel; while the growth factors encapsulated within it are continuously released, accelerating endothelial cell proliferation and migration, and promoting rapid and complete endothelial cell layer coverage. The synergistic effect of the above components enhances the overall biocompatibility of the artificial blood vessel, effectively inhibits thrombosis and intimal hyperplasia, thereby significantly improving the long-term patency rate of small-diameter artificial blood vessels.

[0007] Optionally, the preparation steps of the modified silk fibroin include: mixing silk and sodium carbonate, heating and reacting, filtering, washing, and drying to obtain silk fibroin fibers; mixing the silk fibroin fibers and lithium bromide, stirring in a water bath, dialyzing, and filtering to obtain silk fibroin; mixing silk fibroin and carbodiimide, adding heparin, stirring and reacting in the dark, dialyzing, adding tyrosinase, stirring evenly, and filtering to obtain modified silk fibroin.

[0008] By employing the above-mentioned technical solution, this application uses silk as raw material, and obtains silk fibroin through degumming, dissolution, and purification, and then performs dual functionalization modification on it. Tyrosinase is used to catalyze the cross-linking of tyrosine residues in the silk fibroin molecule, forming a stable three-dimensional network structure within the silk fibroin. This structure not only significantly improves the rigidity and toughness of the silk fibroin matrix itself, but also provides a more stable framework for composites with other components (such as bacterial cellulose), thereby enhancing the overall mechanical properties of the artificial blood vessel inner layer. Carbodiimide is used as a cross-linking agent to covalently graft heparin onto the silk fibroin molecular chain. This provides the artificial blood vessel with durable and highly efficient anticoagulant bioactivity, effectively inhibiting platelet adhesion and thrombus formation in the early stages of implantation.

[0009] Optionally, the growth factors include vascular endothelial growth factor and basic fibroblast growth factor.

[0010] By adopting the above-mentioned technical solution, this application selects a combination of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (BGF). The two work synergistically to construct a highly efficient vascular regeneration microenvironment, which is beneficial to improving the long-term stability and patency of artificial blood vessels. Among them, VEGF can specifically and strongly stimulate the rapid division and migration of endothelial cells, and is a key signal driving the rapid formation of a continuous and complete endothelial cell monolayer, thereby establishing a physiological antithrombotic barrier first. BGF not only helps to promote endothelial cell proliferation and further accelerate the endothelialization process, but its more important role is to simultaneously activate the proliferation of vascular wall supporting cells such as smooth muscle cells and fibroblasts.

[0011] Optionally, the weight ratio of vascular endothelial growth factor to basic fibroblast growth factor is 1:(1-3).

[0012] By employing the above-mentioned technical solution, this application utilizes a specific ratio of vascular endothelial growth factor and basic fibroblast growth factor. This allows for the priority and efficient completion of endothelial layer coverage while simultaneously activating and proliferating supporting cells such as smooth muscle cells, thereby coordinating and promoting the full-thickness, orderly regeneration of the vascular wall. This effectively avoids the pathological risks that may be caused by excessively strong single signals, fundamentally improving the long-term structural stability and functional patency of artificial blood vessels.

[0013] Optionally, the weight ratio of bacterial cellulose to modified silk fibroin is 1:(0.5-1.5).

[0014] By employing the above-mentioned technical solution, this application uses a specific ratio of bacterial cellulose and modified silk fibroin to precisely control the rigidity and elasticity of the composite material, thereby providing a mechanically stable and bioactive microenvironment for cell adhesion, proliferation, and functionalization, comprehensively ensuring that the artificial blood vessel achieves rapid endothelialization, long-term antithrombotic properties, and structural stability. Specifically, bacterial cellulose, through a three-dimensional nanofiber network, provides the composite material with high strength, high modulus, and excellent dimensional stability, effectively resisting the initial impact of blood flow shear forces and preventing lumen collapse. Modified silk fibroin, as an active matrix, contributes excellent flexibility, elasticity, and fatigue resistance through its cross-linked network, enabling the artificial blood vessel to better match the mechanical compliance of natural blood vessels.

[0015] Optionally, the raw materials for preparing the polyurethane layer also include hydroxyapatite and polydopamine.

[0016] By employing the above-mentioned technical solution, polydopamine, acting as a highly efficient "molecular glue," forms a robust coating layer on the surface of hydroxyapatite particles. This structure firstly fundamentally prevents the aggregation of hydroxyapatite particles, allowing them to exist uniformly and stably in a monodisperse state within the polyurethane matrix. Secondly, it significantly enhances the interfacial compatibility and bonding force between the originally hydrophilic hydroxyapatite and the hydrophobic polyurethane matrix. The combined effect of these two factors not only significantly improves the tensile strength and modulus of the polyurethane layer, but more importantly, it greatly enhances its toughness, tear resistance, and long-term fatigue resistance, enabling the vascular stent to better match and withstand cyclic stress under physiological conditions, ensuring long-term structural stability.

[0017] Optionally, the weight ratio of hydroxyapatite to polydopamine is 1:(0.1-0.6).

[0018] By adopting the above technical solution, this application uses a specific ratio of hydroxyapatite and polydopamine, which can ensure that polydopamine fully disperses the role of hydroxyapatite while enhancing the interfacial bonding force, which is conducive to comprehensively improving the mechanical properties, bioactivity and process stability of artificial blood vessels.

[0019] Optionally, the hydroxyapatite has a particle size of 60-80 nm.

[0020] By adopting the above technical solution, this application uses 60-80nm hydroxyapatite, which can enhance biological activity and improve the loading density and fixation efficiency of anticoagulant blood or cell recognition signals, making the biological function of the artificial blood vessel surface more effective and lasting.

[0021] Secondly, this application provides a method for preparing modified silk fibroin artificial blood vessels, employing the following technical solution: A method for preparing a modified silk fibroin artificial blood vessel includes the following steps: S1. Inoculate *Acetobacter xylosiderin* into the fermentation broth, culture, sterilize, wash, and obtain bacterial cellulose; S2. Mix the growth factor mixture with bovine serum albumin and phosphate buffer, then slowly add it dropwise to the modified silk fibroin. Cool down, stir evenly, add the bacterial cellulose and arginine-glycine-aspartic acid polypeptide-modified polycaprolactone, stir evenly, cast into a mold, freeze dry, demold, soak in an ethanol solution containing glutaraldehyde, wash, and dry to obtain the modified silk fibroin composite scaffold. S3. Mix polyurethane and solvent, heat and stir to obtain polyurethane spinning solution; electrospin the polyurethane spinning solution onto a modified silk fibroin composite scaffold, dry to obtain modified silk fibroin artificial blood vessel.

[0022] Optionally, in step S1, the fermentation broth formulation includes: 5 g / L tryptone, 5 g / L yeast extract, 2.7 g / L disodium hydrogen phosphate, 1.5 g / L citric acid, and 20 g / L glucose.

[0023] Optionally, in step S2, the cooling temperature is 1-5℃, the stirring speed is 300-500 r / min, the freeze-drying temperature is -1~-5℃, and the drying temperature is 50-70℃.

[0024] Optionally, in step S3, the solvent is hexafluoroisopropanol, and the heating temperature is 60-80℃.

[0025] Thirdly, this application provides an application of modified silk fibroin artificial blood vessels in vascular replacement surgery.

[0026] In summary, this application includes at least one of the following beneficial technical effects: 1. This application utilizes a polyurethane outer layer, fabricated via electrospinning, to effectively improve the mechanical strength, elasticity, and fatigue resistance of the artificial blood vessel, meeting post-implantation mechanical requirements. The inner layer is a modified silk fibroin composite scaffold, directly in contact with blood. The modified silk fibroin and bacterial cellulose provide a biocompatible substrate, promoting endothelial cell adhesion and growth, thereby reducing abnormal platelet adhesion and activation, and decreasing the risk of early thrombosis. The arginine-glycine-aspartic acid peptide in the composite scaffold specifically recognizes and anchors endothelial (progenitor) cells to the inner surface of the artificial blood vessel; while the growth factors encapsulated within are continuously released, accelerating endothelial cell proliferation and migration, promoting rapid and complete endothelial cell layer coverage. The synergistic effect of these components enhances the overall biocompatibility of the artificial blood vessel, effectively inhibiting thrombosis and intimal hyperplasia, thus significantly improving the long-term patency rate of small-diameter artificial blood vessels. 2. This application utilizes tyrosinase to catalyze the cross-linking of tyrosine residues in silk fibroin molecules, forming a stable three-dimensional network structure within the silk fibroin. This structure not only significantly enhances the rigidity and toughness of the silk fibroin matrix itself but also provides a more stable framework for composites with other components (such as bacterial cellulose), thereby enhancing the overall mechanical properties of the artificial blood vessel inner layer. Furthermore, carbodiimide is used as a cross-linking agent to covalently graft heparin onto the silk fibroin molecular chain. This provides the artificial blood vessel with durable and highly effective anticoagulant bioactivity, effectively inhibiting platelet adhesion and thrombus formation in the early stages of implantation. 3. This application utilizes a combination of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (BGF). The two work synergistically to construct a highly efficient vascular regeneration microenvironment, which is beneficial for improving the long-term stability and patency of artificial blood vessels. Specifically, VEGF specifically and strongly stimulates the rapid division and migration of endothelial cells, serving as a key signal driving the rapid formation of a continuous and intact endothelial cell monolayer, thereby establishing a physiological anti-thrombotic barrier. BGF not only assists in promoting endothelial cell proliferation and further accelerates the endothelialization process, but its more important role lies in simultaneously activating the proliferation of smooth muscle cells and fibroblasts, among other vascular wall supporting cells. 4. The polydopamine of this application acts as a highly efficient "molecular glue," forming a robust coating layer on the surface of hydroxyapatite nanoparticles. This structure fundamentally prevents the aggregation of hydroxyapatite nanoparticles, allowing them to exist uniformly and stably in a monodisperse state within the polyurethane matrix. Secondly, it significantly enhances the interfacial compatibility and bonding force between the originally hydrophilic hydroxyapatite and the hydrophobic polyurethane matrix. These two factors combined not only significantly improve the tensile strength and modulus of the polyurethane layer, but more importantly, substantially improve its toughness, tear resistance, and long-term fatigue resistance, enabling the vascular stent to better match and withstand cyclic stress under physiological conditions, ensuring long-term structural stability. Detailed Implementation

[0027] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0028] This application discloses a modified silk fibroin artificial blood vessel, comprising a modified silk fibroin composite scaffold and a polyurethane layer. The polyurethane layer is electrospun onto the modified silk fibroin composite scaffold. The raw materials for preparing the polyurethane layer include polyurethane. The raw materials for preparing the modified silk fibroin composite scaffold include modified silk fibroin, arginine-glycine-aspartic acid peptide-modified polycaprolactone, bacterial cellulose, growth factors, and glutaraldehyde. The modified silk fibroin bridges the arginine-glycine-aspartic acid peptide-modified polycaprolactone and is loaded with growth factors.

[0029] This application discloses a method for preparing modified silk fibroin artificial blood vessels, comprising the following steps: S1. Inoculate *Acetobacter xylosiderin* into the fermentation broth, culture at 25-30℃ for 5-10 days, sterilize by soaking in sodium hydroxide, wash, and obtain bacterial cellulose; S2. Mix growth factors, bovine serum albumin, and phosphate buffer, then slowly add the mixture dropwise to the modified silk fibroin. Cool the mixture to 1-5℃ and stir at 300-500 rpm for 2-4 hours. Add bacterial cellulose and polycaprolactone modified with arginine-glycine-aspartic acid peptides. Stir at 300-500 rpm for 2-4 hours at 1-5℃. Cast the mixture into a mold and freeze-dry at -5~-1℃. Demold the mixture and soak it in an ethanol solution containing glutaraldehyde. Wash the mixture and dry it at 50-70℃ for 1-2 hours to obtain the modified silk fibroin composite scaffold. S3. Mix polyurethane and solvent, heat and stir at 60-80℃ for 4-8 hours to obtain polyurethane spinning solution; electrospin the polyurethane spinning solution onto the modified silk fibroin composite scaffold, with a spinning voltage of 10-20kV and a feed speed of 4-6mL / h, and dry at room temperature for 24-48 hours to obtain modified silk fibroin artificial blood vessel.

[0030] All raw materials used in the embodiments of this application are commercially available, wherein: Polyurethane, Niron Chemicals (Shanghai) Co., Ltd.; Hexafluoroisopropanol, Shanghai Jizhi Biochemical Technology Co., Ltd.; Acetobacter xylosiders, Shanghai Ruichu Biotechnology Co., Ltd.; Arginine-glycine-aspartic acid peptide-modified polycaprolactone, Xi'an Ruixi Biotechnology Co., Ltd. Vascular endothelial growth factor, Hangzhou Newlong Biotechnology Co., Ltd.; Basic fibroblast growth factor, Hangzhou Newlong Biotechnology Co., Ltd.; Tyrosinase, Hubei Wande Chemical Co., Ltd.; Lithium bromide, Shanghai Aladdin Biochemical Technology Co., Ltd.; Carbodiimide, Shanghai Aladdin Biochemical Technology Co., Ltd.; N-Hydroxysuccinimide, Shanghai Aladdin Biochemical Technology Co., Ltd.; Hydroxyapatite, particle size 60-80nm, Shanghai Aladdin Biochemical Technology Co., Ltd. Glutaraldehyde, Shanghai Aladdin Biochemical Technology Co., Ltd. Specific Implementation

[0031] Preparation Example 1 Preparation of fermentation broth: Mix 5g tryptone, 5g yeast extract, 2.7g disodium hydrogen phosphate, 1.5g citric acid, 20g glucose and 1000mL distilled water, and sterilize by steaming at 121℃ for 15min to obtain fermentation broth.

[0032] Preparation Example 2 Preparation of modified silk fibroin solution: 10g of silk and 500mL of 0.1% w / v sodium carbonate solution were mixed, heated at 100℃ for 60min, filtered, washed, and dried at 50℃ for 3h to obtain silk fibroin fibers; the silk fibroin fibers were mixed with 40mL of 9.3M lithium bromide solution, stirred in a water bath at 70℃ for 3h, dialyzed, and filtered through a 0.22μm microporous membrane to obtain silk fibroin; 3g of silk fibroin, 0.8g of carbodiimide and 0.5g of N-hydroxysuccinimide were mixed, the pH was adjusted to neutral, 0.5g of heparin was added, stirred at 400r / min in the dark for 8h, transferred to a dialysis bag with a molecular weight cutoff of 10kDa for dialysis, 30mg of tyrosinase was added after dialysis, stirred at 400r / min for 8h, and filtered through a 0.22μm microporous membrane to obtain a modified silk fibroin solution with a concentration of 40mg / mL.

[0033] Preparation Example 3 Preparation of polydopamine: 100 mg of dopamine hydrochloride and 100 mL of Tris-HCl buffer solution with pH 8.5 were mixed and placed on a shaker at 200 rpm and incubated at room temperature for 4 h. The solution after reaction was centrifuged at 10,000 rpm for 15 min, the supernatant was removed, the solution was washed with deionized water, dried at 40 °C for 12 h, and ground to obtain polydopamine. Example 1

[0034] At an inoculum size of 5% (v / v), *Acetobacter xylinum* (OD600 = 0.5) was inoculated into 800 mL of the fermentation broth obtained in Preparation Example 1. The mixture was cultured at 28°C for 7 days, then treated in 300 mL of 0.2 mol / L sodium hydroxide solution for 50 min. After rinsing with deionized water until neutral, bacterial cellulose was obtained. 1 mg of basic fibroblast growth factor and 1 mL of phosphate buffer containing 1% bovine serum albumin were mixed and then slowly added dropwise to 250 mL of the broth obtained in Preparation Example 2. In a 40 mg / mL modified silk fibroin solution, the temperature was lowered to 1℃, and the mixture was stirred at 400 r / min for 3 h. 3 g of bacterial cellulose and arginine-glycine-aspartic acid peptide-modified polycaprolactone were added, and the mixture was stirred at 400 r / min for 3 h at 1℃. The mixture was then cast into a mold with an inner diameter of 3 mm, freeze-dried at -1℃ for 15 h, demolded, and soaked in an ethanol solution containing 2% (v / v) glutaraldehyde for 2 h. After washing with deionized water, the mixture was dried at 60℃ for 2 h to obtain a modified silk fibroin composite scaffold. 2 g of polyurethane and 20 mL of 10% w / v hexafluoroisopropanol were mixed and heated and stirred at 70℃ for 5 h to obtain a polyurethane spinning solution. The polyurethane spinning solution was electrospun onto the modified silk fibroin composite scaffold at a spinning voltage of 15 kV and a feed rate of 5 mL / h. The mixture was dried at room temperature for 48 h to obtain a modified silk fibroin artificial blood vessel. Example 2

[0035] At an inoculum size of 5% (v / v), *Acetobacter xylinum* (OD600 = 0.5) was inoculated into 1000 mL of the fermentation broth obtained in Preparation Example 1. The mixture was cultured at 25°C for 10 days, then treated in 400 mL of 0.2 mol / L sodium hydroxide solution for 40 min. After rinsing with deionized water until neutral, bacterial cellulose was obtained. 1.25 mg of vascular endothelial growth factor and 1 mL of phosphate buffer containing 1% bovine serum albumin were mixed and then slowly added dropwise to 300 mL of the fermentation broth obtained in Preparation Example 2. In a 40 mg / mL modified silk fibroin solution, the temperature was lowered to 4℃, and the mixture was stirred at 300 r / min for 4 h. 3.75 g of bacterial cellulose and arginine-glycine-aspartic acid peptide-modified polycaprolactone were added, and the mixture was stirred at 300 r / min for 4 h at 4℃. The mixture was then cast into a mold with an inner diameter of 3 mm, freeze-dried at -3℃ for 12 h, demolded, and soaked in an ethanol solution containing 2% (v / v) glutaraldehyde for 2 h. After washing with deionized water, the mixture was dried at 70℃ for 1 h to obtain a modified silk fibroin composite scaffold. 2.5 g of polyurethane and 25 mL of 10% w / v hexafluoroisopropanol were mixed and heated and stirred at 60℃ for 8 h to obtain a polyurethane spinning solution. The polyurethane spinning solution was electrospun onto the modified silk fibroin composite scaffold at a spinning voltage of 20 kV and a feed rate of 4 mL / h. The mixture was dried at room temperature for 36 h to obtain a modified silk fibroin artificial blood vessel. Example 3

[0036] At an inoculum rate of 5% (v / v), *Acetobacter xylose* was inoculated into 600 mL of the fermentation broth obtained in Preparation Example 1. The mixture was cultured at 30°C for 5 days, then treated in 500 mL of 0.2 mol / L sodium hydroxide solution for 30 min. After rinsing with deionized water until neutral, bacterial cellulose was obtained. 0.75 mg of basic fibroblast growth factor and 1 mL of phosphate buffer containing 1% bovine serum albumin were mixed and then slowly added dropwise to 200 mL of the fermentation broth obtained in Preparation Example 2. In a 40 mg / mL modified silk fibroin solution, the temperature was lowered to 5℃, and the mixture was stirred at 500 r / min for 2 h. 2.25 g of bacterial cellulose and arginine-glycine-aspartic acid peptide-modified polycaprolactone were added, and the mixture was stirred at 500 r / min for 2 h at 4℃. The mixture was then cast into a mold with an inner diameter of 3 mm, freeze-dried at -5℃ for 10 h, demolded, and soaked in an ethanol solution containing 2% (v / v) glutaraldehyde for 2 h. After washing with deionized water, the mixture was dried at 50℃ for 2 h to obtain a modified silk fibroin composite scaffold. 1.5 g of polyurethane and 15 mL of 10% w / v hexafluoroisopropanol were mixed and heated and stirred at 80℃ for 4 h to obtain a polyurethane spinning solution. The polyurethane spinning solution was electrospun onto the modified silk fibroin composite scaffold at a spinning voltage of 10 kV and a feed rate of 6 mL / h. The mixture was dried at room temperature for 48 h to obtain a modified silk fibroin artificial blood vessel.

[0037] The artificial blood vessels obtained in Examples 1-3 were tested for antithrombotic properties, tensile strength, and elongation at break.

[0038] Antithrombotic assay: 200 μL of blood was dropped onto the surface of the artificial blood vessel obtained in this application, followed by 20 μL of 0.2 mol / L calcium chloride solution. The mixture was incubated at 37°C for 10 min, and then 25 mL of deionized water was added. The absorbance of the coating at 541 nm was measured using an ELISA reader. Coagulation index = sample absorbance / standard absorbance × 100%. The standard absorbance is the absorbance at 541 nm of 25 mL of deionized water containing 200 μL of fresh blood.

[0039] Tensile strength and elongation at break testing: The tensile strength and elongation at break of the artificial blood vessels were tested according to GB / T 1039-1992 General Rules for Test Methods of Mechanical Properties of Plastics.

[0040] The test results of Examples 1-3 obtained according to the above test methods are shown in Table 1: Table 1 Performance testing of artificial blood vessels in Examples 1-3

[0041] As shown in Examples 1-3 and Table 1, the coagulation index of the artificial blood vessels in Examples 1-3 is above 93%, the tensile strength is above 10.5 MPa, and the elongation at break is above 234%, indicating that the artificial blood vessels of this application have excellent antithrombotic properties, tensile strength, and elasticity. Modified silk fibroin and bacterial cellulose provide a good biocompatible substrate, which is beneficial to endothelial cell adhesion and growth, thereby reducing abnormal platelet adhesion and activation, and reducing the risk of early thrombosis. Arginine-glycine-aspartic acid polypeptide can specifically recognize and anchor endothelial (progenitor) cells to the inner surface of the artificial blood vessel. Growth factors are continuously released, accelerating endothelial cell proliferation and migration, and promoting rapid and complete endothelial cell layer coverage. The above components work synergistically to improve the antithrombotic properties, tensile strength, and elongation at break of the artificial blood vessel.

[0042] Comparative Example 1 The difference between this comparative example and Example 2 is that the modified silk fibroin in Example 2 is replaced with silk fibroin in this comparative example.

[0043] Comparative Example 2 The difference between this comparative example and Example 2 is that in this comparative example, the arginine-glycine-aspartic acid polypeptide-modified polycaprolactone in Example 2 is replaced with modified silk fibroin.

[0044] The artificial blood vessels obtained in Example 2 and Comparative Examples 1-2 were tested for antithrombotic properties, tensile strength, and elongation at break. The test results are shown in Table 2. Table 2 Performance testing of artificial blood vessels in Example 2 and Comparative Examples 1-2

[0045] As shown in Example 2, Comparative Example 1, and Table 2, the coagulation index of the artificial blood vessel in Example 2 was 96%, the tensile strength was 11.1 MPa, and the elongation at break was 237%, which were significantly better than those in Comparative Example 1. This indicates that grafting heparin onto silk fibroin can improve the anticoagulant activity of the artificial blood vessel and enhance its binding force with bacterial cellulose through tyrosinase cross-linking, thereby improving its mechanical properties.

[0046] As shown in Example 2, Comparative Example 2, and Table 2, the coagulation index of the artificial blood vessel in Example 2 was 96%, the tensile strength was above 11.1 MPa, and the elongation at break was 237%, which were significantly better than those in Comparative Example 2. This indicates that modifying silk fibroin with arginine-glycine-aspartic acid peptides can improve the recognition and anchoring ability of endothelial cells, accelerate the endothelialization process, and thus improve the antithrombotic effect. Example 4

[0047] The difference between this embodiment and embodiment 2 is that in this embodiment, the 1.25 mg vascular endothelial growth factor in embodiment 2 is replaced with 0.625 mg basic fibroblast growth factor and 0.625 mg vascular endothelial growth factor. Example 5

[0048] The difference between this embodiment and Embodiment 2 is that in this embodiment, the 1.25 mg of basic fibroblast growth factor in Embodiment 2 is replaced with 0.417 mg of basic fibroblast growth factor and 0.833 mg of vascular endothelial growth factor. Example 6

[0049] The difference between this embodiment and Embodiment 2 is that in this embodiment, the 1.25 mg of basic fibroblast growth factor in Embodiment 2 is replaced with 0.3125 mg of basic fibroblast growth factor and 0.9375 mg of vascular endothelial growth factor.

[0050] The artificial blood vessels obtained in Examples 2 and 4-6 were tested for antithrombotic properties, tensile strength, and elongation at break. The test results are shown in Table 3. Table 3 Performance testing of artificial blood vessels in Examples 2 and 4-6

[0051] As shown in Examples 2, 4-6, and Table 3, the coagulation index of the artificial blood vessel in Example 2 was 98%, the tensile strength was 12.2 MPa, and the elongation at break was 240%, which were significantly better than those of Comparative Examples 2, 4, and 6. This indicates that the present application uses a specific ratio of basic fibroblast growth factor and vascular endothelial growth factor, which can improve both antithrombotic properties and mechanical properties. Example 7

[0052] At an inoculum size of 5% (v / v), *Acetobacter xylinum* (OD600 = 0.5) was inoculated into 1000 mL of the fermentation broth obtained in Preparation Example 1. The mixture was cultured at 25°C for 10 days, then treated in 400 mL of 0.2 mol / L sodium hydroxide solution for 40 min. After rinsing with deionized water until neutral, bacterial cellulose was obtained. 0.417 mg of basic fibroblast growth factor, 0.833 mg of vascular endothelial growth factor, and 1 mL of phosphate buffer containing 1% bovine serum albumin were mixed and then slowly added dropwise to 300 mL of the broth obtained in Preparation Example 2. In 40 mg / mL modified silk fibroin, the mixture was cooled to 4℃ and stirred at 300 rpm for 4 h. Bacterial cellulose and 3.75 g of polycaprolactone modified with arginine-glycine-aspartic acid peptides were added. The mixture was stirred at 300 rpm for 4 h at 4℃, cast into a mold with an inner diameter of 3 mm, freeze-dried at -3℃ for 12 h, demolded, and immersed in an ethanol solution containing 2% (v / v) glutaraldehyde for 2 h. It was then washed with deionized water and dried at 70℃ for 1 h to obtain a modified silk fibroin composite scaffold. 2.5 g of polyurethane and 25 mL of 10% w / v hexafluoroisopropanol were mixed, and 0.25 g of hydroxyapatite (particle size 60-80 nm) and 0.05 g of polydopamine obtained in Preparation Example 3 were added. The mixture was ultrasonically dispersed at 500 W for 20 min and heated and stirred at 60 °C for 8 h to obtain a polyurethane spinning solution. The polyurethane spinning solution was electrospun onto a modified silk fibroin composite scaffold at a spinning voltage of 20 kV and a feed rate of 4 mL / h. The mixture was dried at room temperature for 36 h to obtain a modified silk fibroin artificial blood vessel. Example 8

[0053] The difference between this embodiment and embodiment 7 is that the weight ratio of hydroxyapatite to polydopamine in this embodiment is 1:0.5. Specifically, the weight of hydroxyapatite is 0.2g and the weight of polydopamine is 0.1g. Example 9

[0054] The difference between this embodiment and embodiment 7 is that the weight ratio of hydroxyapatite to polydopamine in this embodiment is 1:0.6. Specifically, the weight of hydroxyapatite is 0.1875g and the weight of polydopamine is 0.1125g.

[0055] The artificial blood vessels obtained in Examples 5 and 7-9 were tested for antithrombotic properties, tensile strength, and elongation at break. The test results are shown in Table 4. Table 4 Performance testing of artificial blood vessels in Examples 5 and 7-9

[0056] As shown in Examples 5, 7-9, and Table 4, the artificial blood vessel of Example 8 has a tensile strength of 14.3 MPa and an elongation at break of 254%, which is significantly better than that of Examples 5, 7, and 9. This indicates that hydroxyapatite, as a nano-reinforcing phase, can effectively improve the rigidity and strength of the polyurethane layer; polydopamine improves the dispersibility of hydroxyapatite and its interfacial bonding with polyurethane, further enhancing the reinforcing effect and improving tensile strength and elongation at break. This application uses a specific ratio of hydroxyapatite and polydopamine, which can synergistically improve the mechanical properties and long-term stability of the artificial blood vessel.

[0057] The above are all preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. A modified silk fibroin artificial blood vessel, characterized in that, The invention comprises a modified silk fibroin composite scaffold and a polyurethane layer. The polyurethane layer is electrospun onto the modified silk fibroin composite scaffold. The raw materials for preparing the polyurethane layer include polyurethane. The raw materials for preparing the modified silk fibroin composite scaffold include modified silk fibroin, arginine-glycine-aspartic acid peptide-modified polycaprolactone, bacterial cellulose, growth factors, and glutaraldehyde. The modified silk fibroin bridges the arginine-glycine-aspartic acid peptide-modified polycaprolactone and loads growth factors.

2. The modified silk fibroin artificial blood vessel according to claim 1, characterized in that, The preparation steps of the modified silk fibroin include: mixing silk and sodium carbonate, heating and reacting, filtering, washing, and drying to obtain silk fibroin fibers; mixing silk fibroin fibers and lithium bromide, stirring in a water bath, dialyzing, and filtering to obtain silk fibroin; mixing the silk fibroin with carbodiimide, adding heparin, stirring and reacting in the dark, dialyzing, adding tyrosinase, stirring evenly, and filtering to obtain modified silk fibroin.

3. The modified silk fibroin artificial blood vessel according to claim 1, characterized in that, The growth factors include vascular endothelial growth factor and basic fibroblast growth factor.

4. The modified silk fibroin artificial blood vessel according to claim 3, characterized in that, The weight ratio of vascular endothelial growth factor to basic fibroblast growth factor is 1:(1-3).

5. The modified silk fibroin artificial blood vessel according to claim 1, characterized in that, The weight ratio of bacterial cellulose to modified silk fibroin is 1:(0.5-1.5).

6. The modified silk fibroin artificial blood vessel according to claim 1, characterized in that, The raw materials for preparing the polyurethane layer also include hydroxyapatite and polydopamine.

7. The modified silk fibroin artificial blood vessel according to claim 6, characterized in that, The weight ratio of hydroxyapatite to polydopamine is 1:(0.1-0.6).

8. The modified silk fibroin artificial blood vessel according to claim 6, characterized in that, The hydroxyapatite has a particle size of 60-80 nm.

9. A method for preparing a modified silk fibroin artificial blood vessel according to any one of claims 1-8, characterized in that, Includes the following steps: S1. Inoculate *Acetobacter xylosiderin* into the fermentation broth, culture, sterilize, wash, and obtain bacterial cellulose; S2. Mix the growth factor mixture with bovine serum albumin and phosphate buffer, then slowly add it dropwise to the modified silk fibroin. Cool down, stir evenly, add the bacterial cellulose and arginine-glycine-aspartic acid polypeptide-modified polycaprolactone, stir evenly, cast into a mold, freeze dry, demold, soak in an ethanol solution containing glutaraldehyde, wash, and dry to obtain the modified silk fibroin composite scaffold. S3. Mix polyurethane and solvent, heat and stir to obtain polyurethane spinning solution; electrospin the polyurethane spinning solution onto a modified silk fibroin composite scaffold, dry to obtain modified silk fibroin artificial blood vessel.

10. The application of the modified silk fibroin artificial blood vessel according to any one of claims 1-8 in vascular replacement surgery.

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